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Amino-Functionalized 5′ Cap Analogs as Tools for Site-Specific Sequence-Independent Labeling of mRNA
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2017-06-27 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00291 Marcin Warminski 1 , Pawel J. Sikorski 2 , Zofia Warminska 2, 3 , Maciej Lukaszewicz 1 , Anna Kropiwnicka 1 , Joanna Zuberek 1 , Edward Darzynkiewicz 1, 2 , Joanna Kowalska 1 , Jacek Jemielity 2
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2017-06-27 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00291 Marcin Warminski 1 , Pawel J. Sikorski 2 , Zofia Warminska 2, 3 , Maciej Lukaszewicz 1 , Anna Kropiwnicka 1 , Joanna Zuberek 1 , Edward Darzynkiewicz 1, 2 , Joanna Kowalska 1 , Jacek Jemielity 2
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mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5′ cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods: a co-transcriptional labeling method, in which the label is first attached to a cap analog and then incorporated into RNA by in vitro transcription, and a post-transcriptional labeling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labeling of the resulting transcript. After testing the biochemical properties of RNAs carrying the novel modified cap structures, we demonstrated the utility of fluorescently labeled RNAs in decapping assays, RNA decay assays, and RNA visualization in cells. Finally, we also demonstrated that mRNAs labeled by the reported method are translationally active. We envisage that the novel analogs will provide an alternative to radiolabeling of mRNA caps for in vitro studies and open possibilities for new applications related to the study of mRNA fates in vivo.
中文翻译：
氨基官能化的5'帽类似物作为mRNA的特定于位点的序列独立标记的工具
mRNA是蛋白质生物合成的模板，因此，mRNA的运输，翻译和更新是基因表达总体调控的关键要素。随着人们对调节mRNA衰变和定位的机制的兴趣日益浓厚，人们越来越需要能够方便地对mRNA进行荧光标记或亲和标记的工具。我们报告了新的基于mRNA 5'帽类似物的工具，该工具可使用N在帽内进行RNA的位点特异性标记-羟基琥珀酰亚胺（NHS）化学。我们探索了两种互补的方法：共转录标记方法，其中的标签首先连接到帽类似物，然后通过体外转录整合到RNA中；以及转录后标记方法，其中氨基官能化的帽类似物将其掺入RNA，然后化学标记所得的转录物。在测试带有新型修饰帽结构的RNA的生化特性后，我们证明了荧光标记的RNA在脱盖测定，RNA衰减测定和细胞中RNA可视化中的实用性。最后，我们还证明了通过报道的方法标记的mRNA具有翻译活性。
更新日期：2017-06-28
中文翻译：
氨基官能化的5'帽类似物作为mRNA的特定于位点的序列独立标记的工具
mRNA是蛋白质生物合成的模板，因此，mRNA的运输，翻译和更新是基因表达总体调控的关键要素。随着人们对调节mRNA衰变和定位的机制的兴趣日益浓厚，人们越来越需要能够方便地对mRNA进行荧光标记或亲和标记的工具。我们报告了新的基于mRNA 5'帽类似物的工具，该工具可使用N在帽内进行RNA的位点特异性标记-羟基琥珀酰亚胺（NHS）化学。我们探索了两种互补的方法：共转录标记方法，其中的标签首先连接到帽类似物，然后通过体外转录整合到RNA中；以及转录后标记方法，其中氨基官能化的帽类似物将其掺入RNA，然后化学标记所得的转录物。在测试带有新型修饰帽结构的RNA的生化特性后，我们证明了荧光标记的RNA在脱盖测定，RNA衰减测定和细胞中RNA可视化中的实用性。最后，我们还证明了通过报道的方法标记的mRNA具有翻译活性。
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